A QUICK, EFFICIENT, AND COST-EFFECTIVE METHOD FOR ISOLATING HIGH-QUALITY TOTAL RNA FROM TOMATO FRUITS, SUITABLE FOR MOLECULAR BIOLOGY STUDIES

Abstract

Tomato (Solanum lycopersicum L.) is the primary model for the study of fleshy fruits, and research on this species has elucidated many aspects of fruit physiology, development, and metabolism. However, for advancing such studies at molecular biology levels, the RNA isolation from fruit tissues is often essential. The RNA isolation from tomato fruits is complicated because of the presence of high levels of polysaccharides, polyphenolics, pigments, and secondary metabolites and also the varying water content during development. Here, we present an optimized protocol for the isolation of total RNA from the fruit tissues at different developmental stages. In comparison to the previous methods described for the RNA isolation from tomato fruit, this method has the advantages that it does not involve the use of guanidine salts, lyophilizers, and commercial reagents, reduces the time and cost of extraction, overcomes the high water content problem, and promotes RNA quality by inhibiting RNA degradation and minimizing the gDNA, polyphenolic and polysaccharide contaminations. Using this method, high yields of high-purity and intact RNA samples were obtained as confirmed by the spectrophotometric readings and the electrophoresis on denaturing agarose gels. The isolated RNA was employed as a robust template for cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), and temporal gene expression analysis. The functionality of the isolated RNA was further demonstrated through cloning full-length cDNAs encoding β-galactosidase proteins by RT-PCR and sequencing.